U.S. patent number 4,295,199 [Application Number 06/086,917] was granted by the patent office on 1981-10-13 for automatic fluorometer and data processor for performing fluorescent immunoassays.
This patent grant is currently assigned to Bio-Rad Laboratories, Inc.. Invention is credited to Robert E. Curry, Michael G. Simonsen.
United States Patent |
4,295,199 |
Curry , et al. |
October 13, 1981 |
Automatic fluorometer and data processor for performing fluorescent
immunoassays
Abstract
Apparatus is disclosed for quantitating relatively small amounts
of a clinically significant compound which had been fluorescently
tagged. The apparatus has a transparent cell for holding a prepared
liquid sample (which includes fluorescent particles). A light
source generates a stable light beam that is focused on the sample
in the cell so that the beam causes fluorescent emissions by the
particles in the sample. The intensity of the emissions is a
function of the intensity of the light beam and the concentration
of the fluorescent particles in the sample. A detector in optical
communication with the cell receives and senses photons defining
the fluorescent emissions. The number of sensed photons is counted
and the total count from the sample over a fixed time period is a
measure of the number of fluorescent particles in the sample. A
microcomputer system is further disclosed including a primary
microprocessor which is interfaced via latches and buffers on a
data bus to a system memory, system pheripherals, and major system
subassemblies.
Inventors: |
Curry; Robert E. (Novato,
CA), Simonsen; Michael G. (San Rafael, CA) |
Assignee: |
Bio-Rad Laboratories, Inc.
(Richmond, CA)
|
Family
ID: |
22201741 |
Appl.
No.: |
06/086,917 |
Filed: |
October 22, 1979 |
Current U.S.
Class: |
702/21;
250/461.2; 377/10 |
Current CPC
Class: |
G01N
21/6428 (20130101) |
Current International
Class: |
G01N
21/64 (20060101); G01N 033/16 () |
Field of
Search: |
;364/555,413,415,414
;250/461B,458 ;235/92PC,92V ;424/7 ;356/335 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Krass; Errol A.
Attorney, Agent or Firm: Townsend and Townsend
Claims
We claim:
1. A fluorescence immunoassay apparatus for quantitating relatively
small amounts of a clinically significant composition
comprising:
transparent means including a member constructed of a transparent
material and defining therein a hollow cell for holding a liquid
sample including fluorescent particles;
means for flowing the sample into and out of the cell including an
inlet to and an outlet from the cell;
a sample probe;
intake tubing fluidly communicating the probe with the inlet;
discharge tubing fluidly communicating the outlet with a sample
discharge point;
a pump located downstream of the outlet for intermittently flowing
a sample from the probe to the cell, for maintaining the sample in
the cell for a period of time, and for thereafter removing the
entire sample from the cell and the intake tubing and replacing the
removed sample with a fresh sample;
whereby the sample is not subjected to the flow inducing action of
the pump until after it has passed the cell;
a light source for generating a stable light beam focused on the
sample;
whereby the light beam causes fluorescent emissions by the
particles in the sample, the intensity of which emissions is a
function of the intensity of the light beam and the quantity of
fluorescent particles in the sample; and
means in optical communication with the transparent means for
detecting photons resulting from fluorescent emissions by the
particles when excited by the light beam;
whereby the number of photons detected by the photon detecting
means is a function of the number of fluorescent particles in the
sample.
2. Apparatus according to claim 1 wherein the member has a
plurality of mutually perpendicular outer surfaces, one of the
surfaces facing the light source and being perpendicular to the
light beam.
3. Apparatus according to claim 2 wherein the photon detecting
means is positioned to receive fluorescent emissions from the
particles in a direction perpendicular to the light beam; and
wherein a second surface of the member perpendicular to the first
mentioned surface is also perpendicular to the fluorescent
emissions received by the photon detecting means.
4. Apparatus according to claim 3 wherein the photon detecting
means includes a photomultiplier and lens means optically upstream
of the tube for focusing the fluorescent emissions on a
predetermined portion of the photomultiplier.
5. A fluorescence immunoassay apparatus for quantitating relatively
small amounts of a clinically significant composition
comprising:
transparent means for holding a liquid sample including fluorescent
particles;
a light source for generating a stable light beam focused on the
sample;
whereby the light beam causes fluorescent emissions by the
particles in the sample, the intensity of which emissions is a
function of the intensity of the light beam and the quantity of
fluorescent particles in the sample;
means in optical communication with the transparent means for
detecting photons resulting from fluorescent emissions by the
particles when excited by the light beam, said photon detecting
means including signal pulse generating means including a
photomultiplier emitting individual signal pulses for sensed
photons, and discriminator means for eliminating from the output of
the photomultiplier noise pulses having a relatively lesser
amplitude and generated by background noise; and
whereby the number of photons detected by the photon detecting
means is a function of the number of fluorescent particles in the
sample.
6. Apparatus according to claim 4 or 5 including a cutoff filter
optically upstream of the photomultiplier for blocking the light
wavelength with which the fluorescent particles in the sample are
excited from reaching the photomultiplier.
7. Apparatus according to claim 6 including an interference filter
optically upstream of the photomultiplier for removing from light
received by the photomultiplier substantially all light having a
wavelength other than the wavelength of fluorescent emissions.
8. Apparatus according to claim 1 or 5 including stabilizing means
for preventing the intensity of the light beam generated by the
light source from varying by more than a predetermined amount.
9. Apparatus according to claim 8 wherein the stabilizing means
includes a power supply for the light source, means for sensing the
intensity of the beam, and circuit means operatively coupled with
the sensing means and the power supply for adjusting the output of
the power supply in response to changes in the intensity of the
light beam so that the light beam intensity remains substantially
constant.
10. Apparatus according to claim 9 wherein the sensing means
comprises a photodiode, and wherein the circuit means includes
means for amplifying an output of the photodiode, means for
comparing the amplified output of the photodiode with a reference
signal to generate a difference signal, and means for applying the
difference signal to the power supply to correspondingly adjust its
output to the light source.
11. Apparatus according to claim 10 wherein the light source
comprises a tungsten-halogen incandescent lamp and the photodiode
comprises a silicon photodiode.
12. Apparatus according to claim 10 including filter means between
the light source and the sample for conditioning the light beam so
as to minimize light scattering and enhance fluorescent emissions
by the particles, and wherein the photodiode is positioned
relatively proximate to the transparent means and optically
downstream of the filter means.
13. Apparatus according to claim 10 wherein the circuit means
maintains the intensity of the light beam within one percent of a
reference intensity.
14. Apparatus according to claim 5 wherein the transparent means
comprises a member constructed of a transparent material and
defining therein a hollow cell, and including means for flowing the
sample into and out of the cell.
15. Apparatus according to claim 1 or 14 including a band pass
filter disposed in the light beam optically upstream of the member
for removing from the light beam substantially all light other than
light having a wavelength which excites the particles and generates
fluorescent emissions.
16. Apparatus according to claim 15 wherein the band pass filter
eliminates substantially all light from the light beam having a
wavelength other than 490 nanometers.
17. Apparatus according to claim 14 wherein the flowing means
comprises an inlet to and an outlet from the cell, and further
including a sample probe, intake tubing fluidly communicating the
probe with the inlet; discharge tubing fluidly communicating the
outlet with a sample discharge point, and a pump located downstream
of the outlet for intermittently flowing a sample from the probe to
the cell, for maintaining the sample in the cell for a period of
time, and for thereafter removing the entire sample from the cell
and the intake tubing and replacing the removed sample with a fresh
sample; whereby the sample is not subjected to the flow inducing
action of the pump until after it has passed the cell.
18. Apparatus according to claim 1 or 17 wherein at least the
discharge tubing comprises resiliently flexible tubing, and wherein
the pump comprises a peristaltic pump.
19. Apparatus according to claim 1 or 17 wherein the intake tubing
comprises polytetrafluoroethylene tubing.
20. Apparatus according to claim 1 or 17 including first and second
containers for holding samples, and including means for
alternatively fluidly communicating the sample probe with the
containers.
21. Apparatus according to claim 20 wherein the first container
includes a rinse solution; and means for alternatingly fluidly
communicating the sample probe with the first and second containers
in response to each operation of the pump so that at least the
intake tubing and the cell is cleaned with the rinse solution after
the withdrawal of a sample from the intake tubing and the cell and
prior to the replacement of the sample with the fresh sample.
22. Apparatus according to claim 1 wherein the photon detecting
means includes means for generating signal pulses for individually
sensed photons.
23. Apparatus according to claim 22 wherein the signal pulse
generating means comprises a photomultiplier emitting individual
signal pulses for sensed photons, and discriminator means for
eliminating from the output of the photomultiplier noise pulses
having a relatively lesser amplitude and generated by background
noise.
24. Apparatus according to claim 23 or 5 wherein the discriminator
means comprises a source of a reference signal having a lesser
amplitude than the signal pulses, and comparing means receiving as
an input the pulses and the reference signal and generating an
output signal from which pulses having an amplitude which is less
than that of the reference signal have been eliminated.
25. Apparatus according to claim 24 wherein the amplitude of the
reference signal is at least about as large as the amplitude of the
majority of noise pulses.
26. Apparatus according to claim 25 wherein the amplitude of the
reference signal exceeds the amplitude of the noise signals.
27. Apparatus according to claim 22 or 5 including digital pulse
counting means having an input in communication with the signal
pulse generating means for producing a numeric code representative
of the number of pulses received at the input.
28. Apparatus according to claim 27 wherein the pulse counting
means includes an enable input, and further including processor
means coupled to the pulse counting means, the processor means
having means for receiving and storing the numeric code from the
pulse counting means, the processor means also having means for
enabling the pulse counting means for a predetermined interval of
time so that the numeric code stored is representative of
concentration of the clinically significant compound.
29. Apparatus according to claim 28 wherein the processor includes
means for storing the known concentrations of the clinically
significant compound for a plurality of standard samples, means for
storing a corresponding plurality of numeric codes corresponding to
the measured fluorescent activity of the plurality of samples
having known concentration, and arithmetic means for computing a
calibration curve on the basis of the plurality of known
concentrations and the plurality of numeric codes.
30. Apparatus according to claim 29 wherein said arithmetic means
includes means for computing a calibration curve according to at
least two mathematical hypotheses, and for choosing the
mathematical hypothesis that provides the highest correlation
coefficient.
31. Apparatus according to claim 22 or 5 and further
comprising:
digital pulse counting means for receiving the signal pulses from
said photon detecting means and for providing a numeric code output
representative of the number of pulses counted;
programmed microcomputer means in communication with the pulse
counting means, the programmed microcomputer means having means for
selectively enabling the counting means for a predetermined time
interval such that the numeric code from the counting means is
representative of the concentration of the clinically significant
compound, the microcomputer means having means for storing known
concentration information for a plurality of standard samples and
means for accumulating and storing count information corresponding
to said plurality of standard samples, the microcomputer having
means for determining a calibration curve on the basis of the known
concentrations and count information according to at least two
mathematical hypotheses and for selecting the calibration curve
that provides the best correlation coefficient.
32. Apparatus according to claim 31 and further comprising means
for interfacing the microcomputer means to a remote computer for
remote data logging.
33. A fluorescence immunoassay apparatus for quantitating
relatively small amounts of a clinically significant composition
comprising: flow means including a transparent housing defining an
interior cell and an inlet and an outlet to and from the cell,
respectively, for flowing a fluid sample including fluorescent
particles in individual batches through the cell and for holding a
portion of each batch for at least a minimum period of time
stationarily in the cell; optical means including a light source
generating a light beam which is directed into the cell for causing
fluorescent emissions by the particles in the cell, the intensity
of which is a function of the intensity of the light beam and the
quantity of particles in the cell; photosensing means for sensing
the fluorescent emissions over a predetermined time period and for
generating corresponding output signals; means for stabilizing the
intensity of the light beam to prevent variations in the intensity
of the light beam in excess of about one percent from a preset
light beam intensity; discriminator means for eliminating from the
output signals at least a substantial portion of any background
noise signals which are included in the output signals; and
processing means responsive to the output signals for
quantitatively identifying the fluorescent emissions to thereby
measure the number of particles present in the cell.
34. Apparatus according to claim 33 including means defining a
generally L-shaped optical chamber having perpendicular optical
axes; and wherein the cell is disposed at the intersection of the
axes.
35. Apparatus according to claim 33 wherein the optical means and
the photosensing means are disposed in the optical housing and
aligned with the first and second optical axes, respectively.
36. Apparatus according to claim 35 wherein the transparent housing
has a generally square cross-section, the housing including first
and second perpendicular sides which are arranged perpendicular to
the first and second optical axes, respectively.
37. Apparatus according to claim 36 wherein the cell has a square
cross-section and includes first and second perpendicular walls
which are parallel to the first and second sides of the
housing.
38. Apparatus according to claim 33 wherein the photosensing means
comprises a photomultiplier tube, wherein the output signals
comprise signal pulses generated by photons from fluorescent
emissions of the particles in the cell and noise pulses; and
wherein the discriminator means includes means for eliminating the
noise pulses from the output signals before the signal pulses are
fed to the processing means.
39. Apparatus according to claim 38 wherein the signal pulses have
an amplitude greater than the noise pulses, and wherein the
discriminator means includes an amplitude discriminator for
eliminating the noise pulses from the output signals.
40. Apparatus according to claim 33 including pump means in fluid
communication with the outlet and disposed downstream of the cell,
and means for intermittently activating the pump means to flow the
sample out of the cell and to flow a fresh sample into the
cell.
41. Apparatus according to claim 33 wherein the processing means
includes digital counting means, and microcomputer means for
receiving and storing numeric information from the counting means,
the microcomputer means having means for enabling the counting
means for a predetermined length of time so that the numeric
information is representative of the number of particles in the
cell.
42. Apparatus according to claim 41 wherein the microcomputer means
has means for enabling the pump activating means at a time when the
counting means is not enabled so that counting may occur while the
sample is stationary in the cell.
43. Apparatus according to claim 41 wherein the microcomputer means
includes means for storing known concentration information for a
plurality of standard samples and means for accumulating and
storing count information corresponding to said plurality of
standard samples, the microcomputer having means for determining a
calibration curve on the basis of the stored known concentrations
and count information according to at least two mathematical
hypotheses and for selecting the calibration curve that provides
that best correlation coefficient.
44. A fluorescent immunoassay apparatus for quantitating relatively
small amounts of a clinically significant composition comprising:
flow means including a transparent housing having a square
cross-section and including an interior sample cell of a square
cross-section corresponding housing sides and cell walls being
parallel to each other, the housing including an inlet to and an
outlet from the cell; an intake conduit in fluid communication with
the inlet and having an other end; means for alternatingly
connecting the other end of the intake conduit with a container
holding a fluid sample including fluorescent particles and a second
container holding a rinsing fluid; a discharge conduit in fluid
communication with the outlet; pump means disposed downstream of
the outlet and cooperating with the discharge conduit; means for
intermittently activating the pump means to flow a fluid by suction
through the intake conduit, the cell and a portion of the discharge
conduit and for flowing the fluid to a point of discharge;
sequencing means for intermittently activating the pump means and
for alternatingly connecting the intake conduit with the first and
second containers to alternatingly flow sample fluid and a rinsing
fluid into the cell; the sequencing means including means for
activating the pump means for a sufficient length of time to
evacuate the entire fluid in the cell and replace it with fresh
fluid from one of the containers;
an optical housing defining a pair of perpendicular, optical
branches defining perpendicular optical axes, the cell being
disposed in the optical housing and at an intersection of the axes;
a light source in one of the branches for generating a light beam
and first optical means disposed in the optical branch between the
light source and the cell for removing from the light beam
substantially all light other than light of a wavelength which
causes the fluorescent particles to emit fluorescent emissions; a
photomultiplier in the other branch and second optical means for
directing the fluorescent emissions onto the photomultiplier, the
photomultiplier generating a signal pulse for each fluorescent
transmission photon received thereby and noise signals; the housing
including means for preventing light other than fluorescent
emissions caused by the direct excitation of the fluorescent
particles by the light beam from reaching the photomultiplier;
light beam stabilization means operatively coupled with the light
source and including a photosensor disposed in the optical housing
proximate the transparent housing and optically downstream of the
first optical means for sensing the light beam striking the housing
and for adjusting its intensity so that the light beam remains
substantially constant;
discriminator means operatively coupled with the photomultiplier
for removing the noise signals and for generating output signals
comprising substantially only signal pulses;
counting means operatively coupled with the discriminator means for
counting the number of signal pulses emitted by the
photomultiplier; and
microcomputer means having means for enabling the counting means
over a predetermined, constant length of time and for forming an
output which is an indication of the number of fluorescent
particles in the sample cell, the microcomputer means having means
operatively coupled with the pump means for deactivating the pump
means during at least the predetermined length of time and for
activating the pump means during other times so that a tested
sample fluid from the cell is removed, the cell is rinsed with
rinsing fluid, and thereafter a fresh sample fluid is flowed into
the cell.
Description
BACKGROUND OF THE INVENTION
The present invention relates to an automated system for the
immunoassay of subnanogram quantities of certain compositions by
molecular fluorescence.
The quantitative determination of small amounts of clinically
significant compounds, such as metabolites, hormones, drugs and
proteins are of recognized diagnostic importance. Radioimmunoassay
(RIA) has become the standard method for making such determinations
because of its sensitivity and specificity.
However, RIA has certain drawbacks. The radioactivity associated
with RIA may present psychological or physical health hazards to
the technologists, requires special licensing from nuclear
regulatory agencies, requires the special disposal of wastes,
limits the useful life of a reagent kit to a few months at the
most, and requires relatively expensive instrumentation. To
circumvent these drawbacks, alternative methods including
fluorescence immunoassay (FIA) have been developed.
FIA is a technique in which a fluorescent molecule is substituted
for the radioactive label used in RIA. Some of the advantages of
FIA are: no radioactivity, a much longer useful lifetime of the
test components or chemicals necessary for the assay, and
relatively less expensive instrumentation for performing the
assays.
By way of background, the commonly owned co-pending U.S. patent
application bearing Ser. No. 875,475, filed Feb. 6, 1978 for SOLID
PHASE IMMUNOFLUORESCENT ASSAY METHOD, now U.S. Pat. No. 4,201,763,
describes in detail a FIA method for antigens (or haptens) and
their antibodies through the use of an immune reactant related to
the antibody or antigens to be determined which is covalently
bonded or coupled to polymeric particles whose size permits direct
measurement of a labelled immunological reagent's fluorescence in
an aqueous suspension thereof by direct optical spectroscopy. A key
to the method described in that U.S. patent application lies in the
selection of certain types of polymeric particles in sizes which
provide a substantially homogeneous suspension during execution of
the assay. It has been discovered that such a condition exists and
that direct fluorometric measurements can be made when the
polymeric particles have a size of about 0.1-10 microns.
Utilizing such particles, an appropriate immune reactant
immunologically related to unknown antigen or antibody to be
determined is covalently bonded thereto. The particles, unknown
immune reactant, and appropriate fluorescently labelled immune
reactant are mixed under conditions so that a quantity of the
labelled immune reactant proportional to the concentration of the
unknown immune reactant is immunologically bound, directly or
indirectly, to the particles. The particles can then be readily
physically separated and their fluorescence directly measured by
fluorometry.
In accordance with said co-pending U.S. patent application, the FIA
provides water insoluble hydrophilic polymeric particles of about
0.1-10 microns in size and having covalently bonded thereto the
immunological homolog for an antigen or antibody to be determined.
The particles are combined with the antigen or the antibody to be
determined in an aqueous solution to form an immunological bond
therebetween. A fluorescently labelled antigen or antibody
corresponding to the antigen or antibody to be determined is
immunologically bound to the particles. The particles are separated
from the aqueous solution, typically by centrifuging the solution,
and their fluorescence is measured in an aqueous suspension by
fluorometry to obtain information from which unknown antigen or
antibody can be determined.
Any suitable water insoluble polymeric particle may be utilized in
the FIA described in said U.S. patent application. Generally, the
particle will be in spherical or bead form and will be selected
from polymers which can be derivatized to give a terminal primary
amine, terminal carboxyl, or hydroxide group. The antibody or
antigen is then immobilized on the particle under conventional
reaction conditions to produce a covalent bond therebetween. Useful
polymeric particles are formed, for example from crosslinked
polyacrylamides. Other suitable polymeric particles are described
in said U.S. patent application and in the references cited
therein.
After the separation of the beads or particles from the aqueous
solution, their fluorescence is measured in an aqueous suspension
in a fluorometer on a sample by sample basis.
A key to the success of FIA is the reliability and accuracy of the
fluorometer over extended periods of time. In this regard, prior
art fluorometers had certain shortcomings which could affect the
ultimate readout and thus compromise the accuracy of the test.
Conventional fluorometers that operate in an analog mode are
unsatisfactory because of the relative insensitivity of such
fluorometers when measuring the low light intensities encountered
when performing FIA.
Better accuracy can be attained with photon-counting fluorometers
which are relatively simple and inexpensive to construct. Robert E.
Curry et al discuss the construction of photon-counting
fluorometers (hereinafter "fluorometer" unless otherwise indicated)
in "Design and Evaluation of a Filter Fluorometer that Incorporates
a Photon-Counting Detector" on pages 1259-1264 of Clinical
Chemistry, Vol. 19, No. 11, 1973, although the use of such
fluorometers in conjunction with FIA has not heretofore been
considered. The Article notes that photon-counting is an effective
method for minimizing dark current contributions in photomultiplier
tubes since electrons emitted from the dynodes are amplified less
than electrons emitted from the photocathode and level
discriminating circuitry can be used to differentiate between the
dark current and photon signals.
For the determination of small amounts (i.e. from subnanomolar
levels up) of clinically significant compounds by FIA, accuracy
problems are, of course, not fully solved by employing a
photon-counting fluorometer. Stray light, a non-uniform suspension
of the fluorescent beads, light scattering, a variation in the
magnitude of the samples' own fluorescence as well as changes in
the primary light intensity all adversely affect the ultimate
readout and lessen its accuracy. In addition, existing FIA methods
must rely on an essentially manual, sample by sample determination
of the fluorescence which requires the constant attention of highly
skilled and, therefore, costly operators. This in turn has a
tendency to drive up the already high costs for such tests.
SUMMARY OF THE INVENTION
The present invention provides an apparatus for conducting FIA on
an automatic, reliable, self-correcting and continuous basis for
the quantitation of antigens or haptens and antibodies of any
molecular weight and at concentrations from subnanomolar levels
upwards. Initially, in one type of a competitive binding FIA, the
antigen labeled with a fluorescent dye competes with the antigen in
the sample or standard for a limited amount of antibody which is
immobilized on a 0.1-10 micron polyacrylamide bead. After a
suitable incubation, the labeled antigen bound to the antibody
beads is separated from the free fluorescently-tagged antigen in
the supernatant by centrifugation and decantation. After
resuspending the antibody beads in buffer, the fluorescence bound
to the beads is measured in accordance with the present invention
with a fluorometer that utilizes a feedback stabiilized light
source which illuminates a sample in a transparent holding cell to
generate fluorescent emissions. The emissions are sensed by a
photon-counting detector that forms photon generated output pulses
from which background noise is effectively eliminated. System
electronics for the present invention employs large scale
integration microcomputer architecture to provide an automated
capability. In addition to supervisorial and sequencing tasks, the
microprocessor performs data acquisition and data reduction
operations to convert photon count information into antigen
concentration. The apparatus of the present invention assures a
measurement precision and accuracy of about one to three percent
for the above indicated relatively low concentrations being
measured.
The apparatus of the present invention is fully automated, being
capable of processing one sample after the other on a continuing
basis. For this purpose the fluorometer includes a sample cell
which is fluidly communicated with a suitable pump that transports
a predetermined sample volume into the cell, maintains the volume
in the cell until its fluorescence has been measured, and
thereafter replaces the sample in the cell with a new one. The pump
can be operatively coupled with an automatic sample retrieving
unit.
Generally speaking, the present invention provides an apparatus for
quantitating relatively small amounts of a clinically significant
compound such as thyroxine or triiodothyronine which has a
transparent cell for holding a prepared liquid sample. A light
source generates a stable light beam that is focused on the sample
so that the beam causes fluorescent emissions by the particles in
the sample. The intensity of the emissions is a function of the
intensity of the light beam and the concentration of the
fluorescent particles in the sample. A detector in optical
communication with the cell receives and senses photons defining
the fluorescent emissions by the particles when excited by the
light beam. The number of sensed photons is counted and the total
count from the sample (over a given time period) is a measure of
the number of fluorescent particles in the sample.
The associated optics include a lens which focuses the light beam
on the sample, a band pass filter which eliminates substantially
all light from the light beam other than light having a wavelength
which excites the particles and generates fluorescent emissions,
and a heat absorbing filter in the optical branch between the light
source and the sample. The branch of the optics between the sample
and the detector includes a collecting lens, a cutoff filter to
remove the excitation wavelength and thereby the effects of
incoming light scattered by the sample, and a band pass filter
which removes from the light received by the detector substantially
all wavelengths other than the wavelength at which the particles
fluoresce. The sample, the light source as well as the optics are
mounted within a black housing which includes suitable light traps
to prevent light scattering, secondary fluorescent emissions, etc.
As a result, the detector receives substantially only fluorescent
emissions caused by the light beam striking the fluorescent
particles in the sample to assure that the emission, and in
particular the photons of the emissions are the result of
fluorescence caused by the light beam only. This significantly
enhances the accuracy of the photon count by the detector.
To prevent fluctuations in the photon count due to changes in the
intensity of the light beam the light source is stabilized. For
this purpose, a photosensor such as a silicon photodiode is mounted
proximate the sample cell and downstream of the optics in the
branch of the optics between the light source and the cell. A
variation in the light beam intensity sensed by the photodiode is
used to correspondingly increase or decrease the voltage of the
power source for the light source so as to maintain the light beam
intensity constant and at a predetermined level. Consequently,
variations in the light beam intensity due to fluctuations in the
power supply voltage, the age of the light source and the like are
prevented from affecting the fluorescent emissions by the particles
in the sample being tested.
The detector itself is a photomultiplier, preferably a
photomultiplier tube which generates an output charge or signal
pulse in response to each photon sensed by the tube. The
construction of such photomultiplier tubes is well known. Suffice
is to say that the output of the tubes includes noise caused by
thermal electrons emitted by the dynodes of the tube and which
produce corresponding noise pulses at the output side. The noise
pulses have an amplitude significantly lower than the amplitude of
the charge pulses caused by the sensed photons. To prevent the
noise pulses from affecting the ultimate photon count a
discriminator eliminates from the count pulses of an amplitude less
than a predetermined minimum, e.g. less than about the amplitude of
the noise pulses. The photon pulses are passed to appropriate
counting electronics such as cascaded BCD counters.
As a consequence of the foregoing, the ultimate photon count is
highly accurate and is typically within a range of one to three
percent, which is fully within acceptable limits for FIA.
The present invention further fully automates the placement of
samples in the sample cell and their replacement with fresh samples
while assuring a complete rinsing of the cell and associated fluid
conduits to prevent one sample from affecting the photon count of
the next.
In this regard, the present invention contemplates to form a hollow
sample cell in a transparent, e.g. quartz housing having a
generally square (or rectangular) crosssection with corresponding
perpendicular sides. One side faces and is perpendicular to the
incoming light beam and another one faces the photomultiplier and
is perpendicular to the fluorescent emissions received thereby. The
housing includes a sample inlet and a sample outlet connected with
a sample source, e.g. a sample retrieving probe and a sample
discharge point, e.g. a container which receives tested samples as
waste for subsequent disposal. The probe is connected to the cell
housing inlet via an inert conduit such as flexible Teflon tubing.
The outlet of the cell housing is connected to the discharge point
via a readily flexible hose. A pump is located between the outlet
and the discharge point and preferably comprises a peristaltic pump
which conventionally acts on the flexible hose so that the sample
is drawn by suction from the probe into the cell and is not
subjected to the potentially damaging mechanical action of a pump.
Preferably, the sample probe is alternatingly inserted in a sample
holder and a rinse solution holder. In this manner, the sample cell
and the intake tubing are rinsed before a new sample is drawn into
the cell to assure that all remnants of the previous sample are
removed before the fresh sample is introduced.
The present invention further appropriately sequences the operation
of the pump so that a fresh sample is intermittently drawn into the
cell and is stationarily maintained in the cell for the necessary
time period to measure its fluorescence, typically about two
seconds. Thereafter, the tested sample is withdrawn, the cell is
washed with a rinse solution and filled with a fresh sample for
measuring it fluorescence. Thus, the entire process is automated
and the need for constant supervision of the device by a skilled
operator is eliminated.
The microcomputer system of the present invention includes a
primary microprocessor (hereinafter sometimes simply "processor" or
"microprocessor") which is interfaced via appropriate latches and
buffers on a data bus to system memory, system peripherals, and
major system subassemblies. In particular, the primary processor
communicates with the counting electronics, appropriate sequencing
electronics controlling the sample retrieval apparatus, and a
separate arithmetic processor. Additionally, the processor
communicates with keyboard switches, electronics controlling a
display and a keyboard, and an interface that permits data transfer
to a remote computer. The sequencing electronics may itself
comprise a dedicated microprocessor to simplify the primary
processor's communication with the sample retrieval apparatus.
Fluorescence information regarding a particular sample within the
sample cell is accomplished by a counting sequence wherein the
primary processor initially clears the counting electronics and
then enables the counters for a period determined by a software
loop of a fixed number of machine cycles, the machine cycle
duration being precisely fixed by the processor's crystal
controlled clock. Counting is monitored by the processor, and a
multi-digit count is determined and stored in system memory for
subsequent processing.
Prior to counting the fluorescence of actual samples, the
fluorescence of a number of say 10 to 12 standard samples is
measured and the microprocessor computes a doseresponse curve for
one or more and preferably for four data reduction techniques, to
wit a linear interpolation, a logitlog, hyperbolic and reciprocal
program and retains in memory the standard curve parameters for the
reduction technique that gives the best regression coefficient. For
thyroxine or triiodothyonine that coefficient is typically in
excess of 0.990 for the logit function. With the dose-response
curve computed, the fluorescence of each of the samples is measured
and the concentration of antigen in the sample is computed and
printed out. This is accomplished on a continuing basis until all
samples in a given set have been measured.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is an overall, schematic illustration including a more
detailed illustration of the fluorometer of a fluorescence
immunoassay apparatus constructed in accordance with the present
invention;
FIG. 2 is a schematic sample flow diagram including the transparent
sample holding cell constructed in accordance with the present
invention;
FIG. 3 is a schematic of the stabilization circuit for the
fluorometer light source;
FIG. 4 is a schematic of the discriminating circuit of the present
invention;
FIGS. 5A-C are diagrams which illustrate the conditioning of the
output signals of a photomultiplier by the circuit shown in FIG.
4;
FIG. 6 is a schematic of the photon pulse counting circuit of the
present invention;
FIG. 7 is a diagram which illustrates the connection between FIGS.
7A and 7B.
FIGS. 7A-B, taken together, form a simplified circuit schematic of
the microcomputer system of the present invention; and
FIG. 8 is a plan view of the keyboard, illustrating the various key
functions.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Referring first to FIGS. 1 and 2, an apparatus 2 constructed in
accordance with the present invention for performing fluorescent
immunoassays and in particular for quantitating relatively small
amounts of a clinically significant composition such as thyroxine,
for example, comprises a sample holding cell 4 which forms part of
a flow system 6; optics 8 including a first optical branch 10 for
subjecting the sample in the cell to a beam of light and a second
optical branch 12 for collecting fluorescent emissions generated by
fluorescing particles in the sample when excited by the beam; a
photon detector 14 including photon counting electronics 16 which
receives the fluorescent emissions from the sample in the cell; and
system electronics 18 for appropriately analyzing the photon count
for each sample as is more fully discussed below and for sequencing
the various operations performed by the device.
System electronics 18 includes a microcomputer 20, a display
controller 21 for interfacing to a visual display panel 22, a
printer controller 23 for interfacing to a printer 24, a keyboard
26 for inputting various data and commands, and sequencing control
electronics 28 controlled by microcomputer 20 for initiating and
terminating various functions of the apparatus. To facilitate the
understanding of the invention, the subfunctions and subsystems of
the apparatus will be individually discussed before the overall
operation of the apparatus is described in detail.
The sample flow system 6 comprises as its central part a flow
housing 30 constructed of an optically pure, transparent material
such as quartz and which has a square cross-section as is best seen
in FIG. 1 to form four mutually perpendicular sides including a
first side 32 which is perpendicular to the optical axis 34 of the
first optical branch 12 and a second side 36 which is perpendicular
to the optical axis 38 of the second optical branch 12 (and which
is also perpendicular to the first optical axis 34). The housing
defines sample cell 4 which also has square cross-sections and
which has flat interior walls that are parallel to the exterior
sides of the housing. An inlet 40 of the housing is connected to a
length of preferably flexible intake tubing 44 constructed of a
chemically inert material such as polytetrafluoroethylene
(Teflon).
The other end of the intake tubing fluidly communicates with a
sample probe 46. The sample probe may be a hollow, movably mounted
tube (not separately shown) which can be immersed in containers
holding differing solutions. Since the probe does not form a part
of the present invention, it is simply illustrated as comprising a
valve 48 which alternatingly fluidly communicates the intake tubing
44 with a first container 50 holding a sample to be analyzed and a
second container 52 holding a rinsing solution. Additional
containers may be provided to allow a plurality of samples to be
analyzed.
The outlet 42 of the cell housing communicates with a point of
discharge 54 (which may comprise a waste solution bottle or
container, not separately shown) via a discharge hose 56 made of a
readily and repeatedly compressible material, such as Tygon tubing.
Since only waste materials flow through the discharge hose, it need
not be constructed of inert material.
A pump 58 is placed downstream of the sample housing 30 and draws
the sample or the rinse solution from the appropriate container
into the sample cell 4 by suction. Direct contact between the pump
and the fresh sample and potential mechanical damage to the sample
constituents are thereby prevented. Preferably, the pump comprises
a conventional peristaltic pump which engages the outer surface of
the discharge hose 56. Pump 58 is controlled by computer 20 through
appropriate pump driving circuitry 59 which may include a
transistor switch and a relay.
The sample housing 30 is disposed in a generally L-shaped, optical
chamber 60 at about the intersection of the perpendicular chamber
legs 62 and 64, so that the center of the flow cell 4 is at the
intersection of the perpendicular optical axes 34, 38 which also
positions housing sides 32, 36 perpendicular to the respective
optical axes.
The other end of the first optical branch 10 is defined by a light
source 66. Preferably, the light source comprises a 50 W tunsten
halogen lamp provided with a parabolic reflector 68. In the optical
train between the light source and the sample cell are a heat
absorbing filter 70, a condensing lens 72 and a narrow band pass or
interference filter 74. For instances in which the fluorescent is
fluorescein (which has an excitation wavelength of 490 nanometer
(nm) and an emission wavelength of 520 nm) the band pass filter 74
comprises a 490 nm narrow branch pass interference filter.
The second optical branch 12 terminates in a photomultiplier tube
76 (PMT) disposed in the optical chamber leg 64 opposite the second
sample housing side 36. Disposed between the sample housing and the
PMT are a cutoff filter 78 made of low fluorescence glass to block
the excitation wavelength, a collecting lens 80 and a narrow band
pass filter 82. For the above-discussed fluorescent the cutoff
filter is a 515 nm cutoff filter to prevent scattered excitation
light from reaching the PMT while the band pass filter is a 520 nm
narrow band pass interference filter to isolate the emission
wavelength and limit the light reaching the PMT to the emission
wavelength.
To eliminate stray light and light reflections which can directly
or indirectly adversely affect the photon count by the PMT 76, the
interior of housing 60 is black. Further, a light trap 65 is
provided in the form of a well or depression formed in the housing
wall at the extension of the first optical axis 34. The light trap
prevents light which passes through cell housing 34 from being
reflected back into the cell (and thereby causing secondary
fluorescent emissions) and from being reflected into the second
optical branch 12 where it could affect the photon count by the
PMT.
As should be apparent, upon energizing light source 66 light of the
excitation wavelength, e.g. 450 nm is focused onto the sample in
cell 4 and stimulates the fluorescing particles in the samples to
generate fluorescent emissions of a predetermined wavelength, e.g.
520 nm. The fluorescent emissions propagate in all directions
equally and those propagating along the second optical axis 36 are
focused onto the cathode (not separately shown) of the PMT 76. In
the preferred embodiment, the PMT is a nine-stage, side-on type PMT
specifically adapted for photon counting. It operates with a
cascade effect in which an electron emitted by the photocathode due
to an impinging photon is accelerated to the first dynode (not
separately shown) of the PMT by a high voltage bias where it
generates a number of secondary electrons. The number of secondary
electrons is a function of the bias voltage and the dynode material
and structure. The secondary electrons are then accelerated to the
second dynode where each generates a number of additional secondary
electrons. The process continues down the dynode chain to the anode
of the PMT, thereby producing a large current amplification.
Quantitatively, the fluorescent emissions of the excited sample in
cell 4 are a function of the intensity of the excitation light beam
from light source 66 and the concentration of fluorescent particles
in the sample or, since the cell volume is fixed, the total number
of fluorescent particles in the sample cell. Thus, any fluctuation
in the intensity of the excitation light beam would result in a
corresponding change in the intensity of the fluorescent emissions
and cause an error which is a function of the intensity change in
the excitation beam. Since the overall accuracy of the apparatus of
the present invention should not deviate by more than three percent
variations in the excitation beam intensity in excess of one
percent from normal are to be avoided. Light beam intensity changes
in excess of one percent are frequently encountered because of
fluctuations in the line voltage, intensity changes due to the age
of the light source and the like. To prevent these from affecting
the charge pulse count from the PMT 76, the present invention
provides the light source stabilization circuit 84 illustrated in
FIG. 3.
Referring now to FIGS. 1 and 3, a silicon photodiode 86 is
positioned in the optical branch 10 immediately adjacent flow
housing 30, that is optically downstream of heat absorbing filter
70, condensing lens 72 and band pass filter 74, so that light
focused onto the sample cell 4 is also sensed by the photodiode.
The photodiode operates in the photoconductive mode and its output
is amplified in a preamplifier 88, the output of which is compared
to a preset reference voltage V.sub.ref in a comparator 90. The
reference voltage may be obtained from a precision voltage divider
comprising a resistor and a trimmer potentiometer (not separately
shown) and, once set, it is typically not changed. The comparator
output is then used to modulate a triac 92 for the light source to
maintain the intensity of its light beam constant.
In a preferred embodiment of the invention, the comparator senses
the error voltage and forms as an output either +10 V or -10 V,
depending on the error voltage, which is sent to an integrator 94.
The integrator ramps in the appropriate direction to adjust a triac
switching reference voltage. The switching reference is inverted to
obtain both a positive and a negative switching reference. This
bipolar reference is applied to a pair of triac drivers 96 along
with the attenuated 24 V.A.C. output of transformer 98. During each
half cycle of the A.C. wave form, the triac is off until the A.C.
voltage exceeds the switching reference level. When the A.C.
voltage swings above the reference level, the triac is gated on and
conducts until the A.C. voltage falls back below the reference. In
this manner, the duty cycle of the light source A.C. power input is
modulated to maintain its output constant.
With a constant excitation beam the fluorescent emissions for a
given sample in cell 4 remain likewise constant. Referring now to
FIGS. 1, 4 and 5A-C, to maximize the sensitivity of detector 14,
the detector operates in a photon counting mode in which an
individual charge or signal pulse is produced by the PMT 76 for
each photon which reaches the PMT's photocathode (not separately
shown). The PMT operates in this mode for light levels of between
about 10.sup.-11 to 10.sup.-13 W.
Thermal electrons are also emitted by the dynodes and they produce
noise pulses at the anode which constitute an undesirable noise
component which can adversely affect the signal pulse count. The
noise pulses have a substantially lesser amplitude than the photon
generated signal pulses and to eliminate them, a discriminator 100
precedes the photon counting electronics 16. As is illustrated in
FIG. 5A, the noise component of the signal is relatively large at
low voltages and relatively small at higher voltages. To eliminate
most the noise pulses, a reference voltage V.sub.ref is chosen at a
level at which the signal count S is relatively high and the noise
count N is relatively low as is indicated in FIG. 5A.
Both noise and signal pulses are amplified in an amplifier 102 and
the amplifier output is fed to a comparator 104 which acts as an
amplitude discriminator and receives as its second input a preset
reference signal from an appropriate source 106. The comparator
eliminates all pulses having an amplitude less than V.sub.ref as is
indicated in FIG. 5B and thus yields an output signal which
essentially comprises only signal pulses SP while all noise pulses
NP (except for one illustrated in FIGS. 5B and 5C) have been
eliminated.
The reference voltage V.sub.ref of the comparator 104 is typically
preset to provide the optimum signal to noise ratio for the
PMT-amplifier-comparator combination by blocking the majority of
noise pulses while passing most of the charge pulses. If desired,
it can also be continually adjusted by appropriately selecting the
optimal V.sub.ref level at which the signal pulse to noise pulse is
greatest. The output from the comparator, namely the signal pulses
are fed to the counting electronics 16.
Referring to FIG. 6, in a preferred embodiment of the invention,
counting electronics 16 comprises four cascaded four-bit BCD
counters 108, 110, 112, and 114. Each counter has a clock input,
designated CLK, a carry output, designated C/O, and a 4-bit data
output, designated Q. The output from comparator 104 communicates
through a buffer to the input of counter 108; the carry output of
counter 108 communicates through a buffer to the input of counter
110; the carry output of counter 110 communicates through a buffer
to the input of counter 112; the carry output of counter 112
communicates through a buffer to the input of counter 114; and the
carry output of counter 114 communicates to the clock input of a
"carry" flip-flop 120. Flip-flop 120 has a clear input, designated
CLR which may receive signals from microcomputer 20 on a line 121.
The output of flip-flop 120, designated Q, communicates to
microcomputer 120 via a line 122. The outputs of counters 108 and
112 are communicated to the inputs of an 8-to-4 multiplexer 124.
Similarly the outputs of counters 110 and 114 are communicated to
the inputs of an 8-to-4 multiplexer 125. The 4-bit outputs of
multiplexers 124 and 125 together form an 8-bit data line 126.
Multiplexers 124 and 125 have respective select inputs, designated
SEL, which are tied together and receive signals on a line 127.
Each counter also has an enable input, designated EN, to enable
counting, and a clear input, designated CLR, to zero the counter
contents. The enable inputs are tied together and receive signals
from microcomputer 20 on a line 128. The clear inputs are tied
together and receive signals on a line 129.
FIGS. 7A and 7B, taken together, form a system block diagram of
system electronics 18 that controls the operation of the present
invention. A table of preferred component types will be set forth
below. The central component of the system electronic circuitry is
a primary microprocessor 130 which interfaces to system memory,
peripherals, and other subassemblies as will be described below.
Microprocessor 130 is driven by a 4 MHz crystal clock 131.
Microprocessor 130 communicates to other portions of the
electronics via a 12-bit address bus 132 (designated AD0-AD11) and
an 8-bit system data bus 135 (designated DB0-DB7). Data bus 135 is
time multiplexed so that it carries high order address and status
information during a first portion of the microprocessor execution
cycle and data during the latter portion of the cycle. The high
order address information (designated AD12-AD15) is latched at a
quad-D flip-flop 137 by the NADS processor strobe and used to drive
a 4-to-16 decoder 140 for selecting a particular device (memory
unit, peripheral, etc.) for input or output. Some of the decoder
outputs are used as strobes to actuate various system functions
while other strobes originate directly at microprocessor 130. In
addition, a number of logic gates, not shown, are used to
coordinate such strobes with other status information in order to
achieve correct device selection and control. Thus, address bus 132
provides microprocessor 130 with 12 dedicated address lines
(designated AD0-AD11) which, in conjunction with the 4 bits latched
by flip-flop 137, provide a total of 16 address lines.
Microprocessor 130 has self-contained inputs and outputs for
control of peripheral devices. The outputs consist of three flags
(designated FL0, FL1, FL2) and a serial port intended for serial
data communication. The inputs consist of two sense inputs
(designated S.sub.A and S.sub.B) and a serial in port which is not
used. Microprocessor 130 is interfaced to an arithmetic processor
145 by two quad D input latches 147 and 148 and a 6-bit tri-state
output buffer 150. The interface is a synchronous. Arithmetic
processor 145 provides an interrupt signal on a line 152 to sense
input S.sub.A of microprocessor 130 in order to allow
microprocessor 130 to respond to arithmetic processor 145 during
execution of a mathematical operation. One of the outputs of latch
148 communicates to pump driving circuitry 59 for activating pump
58.
System memory comprises random access memory units 160 and 162
(hereinafter sometimes RAM's) that together provide 1024 8-bit
bytes of read/write memory, and read-only memory units 165, 167,
168, and 170 (hereinafter sometimes ROM's) that together provide
8192 bytes of read-only memory. The system memory units are coupled
directly to address bus 132, and are coupled to a memory interface
data bus 172 which is coupled to system data bus 135 through
bidirectional tri-state buffers 175 and 177, the operations being
controlled by the NRDS and NWDS strobes of microprocessor 130. Bus
172 also communicates to sequencing circuitry 28 to allow
microprocessor 130 to control sample retrieval. Sequencer 28 is
selected by appropriate control lines from decoder 140 and
associated logic gates in addition to the NWDS and NRDS strobes.
Sequencing circuitry 28 may itself by microprocessor controlled.
Bus 172 also connects to an IEEE 488 interface card 179 which
allows the apparatus of the present invention to communicate to a
central computer which could accumulate data from multiple
instruments for statistical or other purposes.
Microprocessor 130 is interfaced directly to photon counting
electronic circuitry 16 via 1-bit data lines 121, 122, 127, 128,
and 129, and 8-bit data line 126. Line 126 carrying counter data is
coupled to processor data bus 135 while carry line 122 is coupled
to sense input S.sub.B. Counter clearing line 121 and carry
clearing line 129 are coupled to respective outputs of decoder 140,
count enabling line 128 is coupled to flag FL0, and data select
line 127 is coupled to address line AD0 of address bus 132. Thus,
the numerical contents of the BCD counters and the state of the
carry flip-flop may be read by microprocessor 130, and signals may
be sent in order to clear the counters, clear the carry flip-flop,
and enable the counters.
Microprocessor 130 communicates with display 22 through appropriate
multiplexing circuitry in display control circuitry 21.
Microprocessor 130 and display controller 21 are synchronized by a
7.8 kHz derivative of microprocessor clock 131 provided by a divide
by 512 network 200 which drives the display multiplexing circuitry.
Display 22 is capable of displaying 16 5.times.7 dot matrix
alphanumeric characters which are refreshed at the rate
approximately 70 Hz. Data for the display, consisting of 16
sequentially output ASCII characters, is stored in RAM's 160 and
162 and subsequently read out onto data bus 135 and latched by
microprocessor 130 at a latch 205. The display circuitry then
accesses this data by strobing a count update line 207 to transfer
the data latched at latch 205 to a latch 210. Blanking of the
display is controlled by a flip-flop 212 which in turn is
controlled by strobes from decoder 140.
Microprocessor 130 interfaces a printer 24 through appropriate
printer controller circuitry 23. The printhead of printer 24 is an
impact dot matrix type utilizing seven solenoid actuated strikers
and a motor-driven carriage to move the solenoids across the paper.
Paper feed is accomplished by a feed solenoid and friction drive to
provide a print speed of 2.3 lines per second. The printer control
circuitry is preferably microprocessor based and receives ASCII
input data into a 20-position buffer and then controls the motor
drive and solenoid firing to form the corresponding characters. Six
data lines for ASCII characters, a "print out" line and a "feed
out" line for printer control circuitry 23 are coupled to the
output of latch 205.
FIG. 8 is a plan view illustrating keyboard 16. Keyboard 26
consists of an array of 28 printed circuit mountable switches
arranged as seven columns by four rows. The switches are of the
mechanical contact type and are hermetically sealed at the front
panel of the instrument by a rubber gasket to prevent leakage of
spilled fluids into the instrument. The keyboard switches are used
by the operator to enter assay parameters, standards
concentrations, and to execute system commands. In particular,
keyboard 16 comprises 11 numeric keys 230 (0-9 and decimal point),
a clear key 232, 8 concentration units keys 235 (mg/cl, .mu.g/dl
ng/dl, %, miu/ml .mu.iu/ml, ng/ml, and pg/ml), four mathematical
reduction keys 237 (linear interpolation, reciprocal, hyperbolic,
and logit-log), and four system command keys consisting of an ENTER
key to 240, a RUN key 242, a PUMP key 245, and a FEED key 247. A
"reset" switch is mounted at a separate front panel location and
communicates to reset circuitry 250 for providing a reset pulse to
microprocessor 130. Two high order lines of address bus 132
communicate to a decoder 252 which supplies four output lines
corresponding to the four rows. Seven lines corresponding to the
seven columns communicate via a tri-state buffer 255 to processor
data bus 135. Thus, the keyboard is scanned by sequentially
addressing each keyboard row with a low strobe from decoder 252.
Presence of a closed keyboard switch is detected by enabling buffer
255 and reading the keyboard columns. A low on a given line into
buffer 255 indicates closure of a switch in the corresponding
column while the specific row of that column is determined by the
status of the two address lines input to decoder 252.
While many types of integrated circuit components could be used,
preferred components are set forth in the following table,
designated Table I.
TABLE I ______________________________________ Reference Numeral
Description ______________________________________ 102 OP AMP
BI-FET LH0062CH 104 Comparator LM360H 108 Decade Counter 74LS160N
110 Decade Counter 74LS160N 112 Decade Counter 74LS160N 114 Decade
Counter 74LS160N 120 JK Flip-flop 74LS73N 124 Quad 2-Data
Selector/Multiplexer 74LS257N 125 Quad 2-Data Selector/Multiplexer
74LS257N 130 Microprocessor 8-bit INS8060 137 Quad Latch 74LS75N
140 4-to-6 Decoder/Demultiplexer 74LS154N 145 Processor 6-bit
MM57109 147 Quad Latch 74LS75N 148 Quad Latch 74LS75N 150 Hex
Buffer 74LS365N 160 1024x4K Static RAM P2114 162 1024x4K Static RAM
P2114 165 2048x8 EPROM B2716 167 2048x8 EPROM B2716 168 2048x8
EPROM B2716 170 2048x8 EPROM B2716 175 Octal Buffer 81LS95N 177
Octal Buffer 81LS95N 205 Latch 74LS273N 210 Latch 74LS273N 212 JK
Flip-flop 74LS73N 252 Decoder 74LS138N 255 Octal Buffer 81LS95N
______________________________________
The software for the operation of microprocessor 130 resides in
read-only memory units 165, 167, 168, and 170, and is organized
into two major subsets. The first subset comprises operating
procedure instructions including a main program for executing a
standard assay sequence and subroutines for scanning keyboard 26,
strobing display 22, operating printer 24, performing the count
sequence, and operating pump 58. The second subset comprises data
reduction procedures to be described in detail below.
The standard assay sequence includes a calibration sequence and a
sample sequence. Prior to counting the fluorescence of actual
samples, the fluorescence of a number of standard samples of known
concentration is measured and a dose response curve is calculated.
Thereafter, for the measured fluorescence of a sample, the antigen
concentration is calculated and printed out.
Turning first to the operation of the device with respect to
acquiring the fluorescence count for a given sample (standard or
actual), the operation of the device may be described as follows.
To briefly summarize it, the operation commences with computer 20
instructing sequencer 28 to operate valve 48 to connect the intake
tubing 44 with sample container 50. The computer then energizes
pump 58 long enough so that a sufficient volume of the sample from
the container 50 is drawn (by vacuum) through the intake tubing 44
to completely fill sample cell 40 in sample housing 30. The
computer than de-energizes the pump.
With the light source 66 energized, the fluorescent particles in
the sample cell 4 fluoresce and emit fluorescent emissions some of
which are directed onto PMT 76. Photodiode 86 and stabilizing
circuitry 84 maintain the intensity of the light beam from source
66 constant.
The fluorescent emissions received by the PMT result in a
bombardment of the PMT cathode (not separately shown) by photons
with each photon resulting in a charge or signal pulse. The
discriminator circuit 100 removes from the output of the PMT
relatively low amplitude noise signals so that the output from the
discriminator circuit comprises substantially only signal pulses
with each pulse representing a fluorescent emission photon received
by the PMT cathode. These pulses are fed into BCD counter 108.
Microprocessor 130 commences the counting sequence by sending low
going strobes on carry clearing line 121 and counter clearing line
129 in order to clear the counters and the carry flip-flop.
Microprocessor 130 then sets FL0 high in order to communicate a
high signal on counter enabling line 128 to enable the counters and
begin the count period. The duration of the count period is
determined by a software loop of a fixed number of machine cycles,
the machine cycle time being precisely fixed by crystal controlled
clock 131. During the count period, microprocessor 130 periodically
checks carry flip-flop 120, and if a carry occurs, the
microprocessor increments a carry storage register in memory and
clears the flip-flop. At the end of the count period, FL0 is set
low to stop the count. Microprocessor 130 then reads this data
through multiplexers 122 and 125 and adds it (4 BCD digits) to the
number of carries multiplied to 10,000 to obtain the total number
of counts during the count period. Counting continues for an exact,
constant predetermined length of time, say two seconds as
determined by the processor clock. At the end of the two second
time period, counting is discontinued and the total count is
further processed as is described below.
During or immediately after the photon count, sequencer 28 switches
valve 48 of the sample probe 46 so as to connect the intake tubing
44 with rinse solution container 52. Upon the termination of the
photon count computer 20 energizes pump 58 again which now draws
rinsing solution into the intake tubing. The pump remains energized
until at least the entire sample cell 4 is filled with rinsing
solution. Drawing the rinsing solution into the sample cell causes
the discharge of a corresponding volume of already tested sample
fluid through the discharge hose 56 to discharge point 54 and, for
example, into an appropriate waste fluid container (not shown).
Once the sample cell is filled with rinsing solution, valve 48
again fluidly connects the sample container 50 with intake tubing
44. Pump 58 may continue to operate or may be de-energized until
the operation of the valve has been completed. Thereafter, the pump
draws a fresh sample into the sample cell 4 as above-described,
while a corresponding volume of the rinsing solution is discharged
via the discharge hose. Of course, the original sample in container
50 will have been replaced with a fresh sample or a new sample
container will have replaced the original one unless it is desired
to test the same sample twice.
Having described the operation of aspirating and measuring the
fluorescence of a given sample, the overall operation of the
apparatus by the operator may be understood. Operation of the
apparatus in order to carry out a standard assay procedure is best
understood with prior reference to the key functions of the various
keys illustrated in FIG. 8.
Numeric keys 230 are used to key in numeric assay parameters
(precision and multiplicity) and standards values. The maximum
number of digits or alphanumeric characters which may be entered is
14. The numerial "1" key performs a special function which allows
the user to halt the system during the run. In order to achieve
this function the user must depress and hold the numerial "1"
during printout of either the standards results or a sample result.
The system scans the keyboard immediately following the printout
sequence. The system remains stopped until RUN key 242 is
depressed.
CLEAR key 232 is used to erase values from the system memory, if a
keying error is made. When CLEAR key 232 is depressed and released,
the display and corresponding active location in system memory are
cleared to 0. Number entry may then be corrected by depressing the
appropriate numeric and concentration keys. Clear key 232 may also
be used in a memory modification procedure to delete a bad point
from the standard curve by clearing the corresponding data from the
system memory.
Concentration units keys 235 are used to key in concentration units
during entry of standards values. When the user depresses the key
labeled with a particular concentration unit appropriate for the
assay, previously entered numerical values are left shifted by six
spaces in the display unit, and the selected concentration units
are stored in the rightmost six spaces. The concentration unit is
stored in system memory.
Data reduction keys 237 are used to let the user select a data
reduction mode in the event that automatic data reduction selection
is not desired.
ENTER key 240 is used to enter assay parameter and standard values
in system memory. After an assay parameter or standard value has
been keyed into the display using numeric keys 230 and
concentration keys 235, it may be entered to memory by depressing
ENTER key 240. This causes the display value to be entered into
memory and printed. This also has the effect of incrementing the
memory location pointer so that the next memory storage location
becomes available for another value to be keyed in and entered. The
ENTER key is also used to access memory locations for modification
of values or clearing of a location which is to be deleted.
RUN key 242 is used to command the system to begin loading of
standards and/or samples. Pressing this key after entry of all
standards causes the system to display the instruction "LOAD
STANDARDS" and pressing the key again causes the system to begin
aspirating and counting standards and samples. If the system is
halted during the assay by use of the numeric "1" key, the run may
be continued by pressing RUN key 242.
PUMP key 245 provides a start/stop function for pump 58. One press
of the key starts the pump, a second press stops the pump. This key
is used to run the pump for the purpose of flushing the system.
FEED key 247 causes the printer to feed the paper forward when the
key is depressed to enable the user to format the printout.
Having described the above key functions provided by the system
software, the operation of the instrument during a standard assay
procedure may now be understood. Initially, test tubes containing
the standards and the samples to be assayed are loaded into the
sample holder for subsequent sequencing. In particular, the samples
are loaded so that the standards are measured first for
calibration, and the samples to be assayed are measured next.
During the initialization, the message "ENTER PRECISION" is
displayed on display 22. The operator responds to this prompt by
entering via numeric keys 230 the desired allowable percentage
deviation of replicates from one another. After the desired value
has been keyed in, the operator pushes ENTER key 240, at which
point the system prints out the entry and provides an "ENTER #
REPLICATES" prompt. The operator responds by keying in either 1 or
2 which will cause samples to be calculated singly or in pairs
where pairs of samples are treated as duplicates so that their
agreement may be checked. The operator then presses ENTER key 240,
and is provided with an "ENTER STANDARD" prompt.
In response to the "ENTER STANDARD" prompt on display 22, the
operator enters the concentrations of the standards into system
memory in the order in which they were loaded onto the sample
holder. Each concentration is keyed in, and upon verifying the
entry on the display, the operator presses "ENTER" key 240 to store
the value in memory and increment the memory pointer. A maximum of
16 standard entries is typical. Upon entry of a given standard
concentration, the entry is printed out as it is stored, so that
after all standards are entered, the operator may check the print
out to ensure that the values are correct. The system provides the
operator with the ability to correct an incorrect entry.
After the operator has entered and verified all standard
concentrations, he presses RUN key 242 in response to which display
22 provides a "LOAD STANDARDS" prompt. The operator verifies that
the first test sample (in container 50) of the standard set
communicates with intake tubing 44 and upon verification, presses
RUN key 242 again. The system now aspirates and counts all of the
standard samples. After counting the fluorescence from the
standards, the system prints out the concentrations and
corresponding counts for all standards. In the event that
non-automatic data reduction is desired, the operator halts the
system by depressing and holding the numeral "1" key during the
print out. In this mode, the operator then selects one of the four
data reduction routines by depressing one of data reduction keys
237 corresponding to the four routines. In general, the routine
giving the highest correlation value should be used. The operator
may eliminate a bad standard sample from the calculation by
modifying the memory locations to zero out the concentration of the
standard to be eliminated. If automatic data reduction is desired,
the system automatically performs the data reductions by running
the reciprocal, hyperbolic, and logit-log routines and determining
which provides the highest correlation coefficient.
After the calibration parameters have been determined, the system
aspirates and counts the samples sequentially as above discussed,
and subsequently prints out the counts, the sample number, and the
calculated sample concentration for each sample.
With respect to the second subset of the system software relating
to data reduction procedures, such software provides the capability
for microprocessor 130 to run in tandem with arithmetic processor
145 to execute standard curve fitting routines and calculate and
evaluate results. In all, four curve fitting procedures are
available--namely reciprocal, hyperbolic, logit-log, and linear
interpolation. In all cases, the routine has as input a set of
points [(u.sub.i,v.sub.i) i=1,n] where u.sub.i is the concentration
of the i.sup.th standard sample and v.sub.i is the number of counts
for the i.sup.th standard sample.
In each of the first three procedures, the data is transformed to a
new set of data points [(x.sub.i,y.sub.i) i=1,n] and a
least-squares fit of the transformed data to the functional form
y=Ax+B is carried out. This is a straight line of slope A and
intercept B. The slope A, the intercept B and the correlation
coefficient R are calculated as follows: ##EQU1## where:
##EQU2##
For the reciprocal procedure, the transformation is:
x.sub.i =u.sub.i
y.sub.i =1/v.sub.i
so that the relationship between concentration u and counts v is
1/v=A u+B.
For the hyperbolic procedure, the transformation is:
x.sub.i =ln u.sub.i
y.sub.i =ln v.sub.i
so that the relationship between concentration u and counts v is ln
v=A ln u+B, or equivalently, v=C u.sup.A where ln C=B.
For the logit-log procedure, the transformation is: ##EQU3## where
b.sub.0 is the count for a zero dose. Thus, in order to execute
this procedure, a zero standard (b.sub.0) must be entered and
counted. The zero standard counts must be greater than the counts
for the other standards and samples in the set. If a given standard
sample has counts greater than b.sub.0 (the zero standard), the
point will be eliminated from the standard's data automatically. In
the event that a sample being assayed gives counts greater than
b.sub.0 and logit data reduction is being used, the sample will be
flagged and the display will show "UNDER RANGE".
Once a given curve has been fitted, the calculated values of
concentration are calculated using the curve fit parameters. A
check on standard curve validity is accomplished by requiring the
computed correlation coefficient to exceed some prescribed minimum.
If this condition is not met, the system will halt all
operations.
It can thus be seen that the goodness of the fit depends on how
well the particular transformation linearizes the data. While the
above three procedures provide very good fit for many types of
assay, there are certain situations where the transformed data
points do not fit a straight line to the required precision. In
such a situation, the operator may instruct the computer to use a
linear interpolation procedure in which the curve is fitted is a
sequence of straight line segments joining adjacent points. Such a
fit is not actually a fit since the curve is guaranteed pass
exactly through all the data points.
After the particular data reduction routine to be applied to the
samples has been determined (automatically on the basis of the
highest correlation coefficient, or manually by the operator), the
slope and intercept parameters for the selected routines are
computed and stored in memory. Thereafter, when samples are assayed
and their counts determined, concentrations are calculated using
these parameters.
* * * * *